Ischemic mitral regurgitation (IMR) is a common complication that doubles mortality and increases heart failure after myocardial infarction (MI). Effective repair has been elusive for IMR, which is caused by left ventricular (LV) remodeling that tethers the mitral valve (MV) leaflets and restricts their closure - a mismatch between valve and LV size. Late-stage valves are also stiff and fibrotic, further limiting effective closure. Standard therapies assume valve size is fixed, but valves have the potential for cellular activation, and flexible enlargement of the tethered MV could reduce IMR. Valve adaptation can be affected by mechanical stretch, the ischemic milieu, and MR turbulence. We therefore developed a large-animal model to vary these factors independently using 3D echo to follow MV area noninvasively, correlated with cellular and molecular studies. In that model, mechanical tethering induced by papillary muscle traction short of producing MR increases MV area and thickness over two months with reactivated endothelial-mesenchymal transformation (EMT), a developmental process. Adding a distal apical MI (limited apical LV remodeling) to mechanical tethering over two months markedly increases EMT, with expression of pro-fibrotic transforming growth factor (TGF)-, endothelial activation (VCAM-1), collagen deposition, and infiltration of CD45+ cells. Blood-borne wound- healing CD45+ cells create sclerosis of other organs by differentiating into collagen-producing myofibroblasts. We will therefore test the central hypothesis that early compensatory MV growth mechanisms in the IMR setting later become decompensatory, leading to stiffness that increases MR. Aim 1 will correlate fibrosis and stiffness with TGF- expression, endothelial activation and CD45+ cell infiltration at 2, 6 and 10 months in models of MI+tethering and the clinical-type scenario, inferior MI. Aim 2 will isolate the MV CD45+ cells and test whether they have the characteristics of fibrocytes, circulating myofibroblast precursors; MV and peripheral blood CD45+ cells will be tested for adhesion to MV endothelial cells stimulated by MI-released cytokines, differentiation into myofibroblasts, and possibly influencing native MV cells to undergo similar pro-fibrotic change. Aim 3 is based on preliminary studies that Losartan, a TGF- inhibitor, reduces EMT, CD45+ cells, endothelial activation and MV thickening at two months in the tethering+MI model; in contrast, those findings persist when LV remodeling is comparably reduced by mechanical LV constraint. Losartan also inhibits TGF- -mediated EMT in vitro. We will test whether Losartan, unlike LV constraint, reduces long-term pro-fibrotic events from 2 to 6 and 10 months in the tethering+MI model, and study downstream TGF- signaling, recently shown to have therapeutic implications. This proposal combines investigators with complementary strengths in physiologic modeling and imaging, MV histopathology, endothelial cell biology and biomechanics. It addresses unmet clinical needs in a common disease, aiming to increase our understanding of MV adaptation. It begins to test potential therapies that, if corroborated, could be rapidly translated to reduce IMR in patients. PUBLIC HEALTH RELEVANCE: Mitral regurgitation (MR) is a frequent, difficult to repair complication of myocardial infarction (MI) that doubles heart failure and mortality; it is caused b tethering (stretch) of the mitral valve by damaged and bulging heart walls that prevents valve closure, and is compounded by late valve stiffening. Until recently, mitral valve size has been viewed as fixed in this setting, but valve growth in adaptation to tethering could reduce this regurgitation if valve flexibility can be maintained. In this project, an interdisciplinary team wil use a new model to fill this unmet clinical need in a common disease by increasing our understanding of how adaptive valve processes can become maladaptive, and will begin to test new therapies to reduce this complication that, if corroborated, can be rapidly translated to benefit patients.